MDSCs定向分化的调控及其在压力性尿失禁中作用的实验研究
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摘要
女性压力性尿失禁(stress urinary incontinence, SUI)是指由于各种原因引起盆底肌肉筋膜组织松驰,膀胱和尿道解剖位置改变及尿道阻力降低,致使排尿自禁功能障碍,从而在腹压突然增高时尿液不自主流出的一种疾病。SUI是临床常见疾病,23%～44%的妇女有尿失禁症状,随人口老年化加重,其发病率会不断升高,不仅严重影响患者的生活质量,而且会加重社会医疗负担。
导致SUI的关键机制主要有两种,尿道固有括约肌缺陷(intrinsic sphincter deficiency,ISD)和膀胱颈支撑功能障碍。ISD是导致女性SUI的重要原因,传统的治疗方法对于ISD所致的SUI的疗效差。从本质上改善ISD的方法是纠正其成肌功能障碍,即恢复ISD所致SUI患者成肌细胞功能。随着组织工程和基因治疗发展及干细胞研究深入,干细胞移植成为肌肉组织损伤修复的重要手段,这也为ISD型压力性尿失禁的治疗带来了希望。
肌源性干细胞(Muscle derived stem cells,MDSCs)是高度未分化的多潜能细胞,在适当条件下,MDSCs可分化为骨骼肌成肌细胞、造血细胞、成骨细胞、成软骨样细胞。研究发现骨骼肌损伤时,骨骼肌干细胞可分化形成肌纤维参与肌损伤修复过程。将MDSCs注入心肌梗死或尿失禁模型大鼠,MDSCs可参与心肌或尿道平滑肌的修复。在人体,已有成功用MDSCs治疗心肌梗死或压力性尿失禁的的研究报道。
目前MDSCs向具体细胞分化的机制尚不完全明确,可能与移植后细胞所处环境中的某些信号有关,不同的微环境可诱导MDSCs向不同的细胞分化,即MDSCs具有组织分化特异性。但是在MDSCs移植治疗骨骼肌损伤的研究中发现,MDSCs除分化为骨骼肌细胞外,在TGF-β等细胞因子的刺激下还可分化为成纤维细胞,导致骨骼肌纤维化,而影响细胞移植的疗效。在MDSCs移植治疗压力性尿失禁的研究中也发现:MDSCs或成纤维细胞移植到中段尿道周围,对尿失禁的治疗效果是相同的,提示:MDSCs移植后可能转分化为成纤维细胞参与尿道修复。
我们推测:肌源性干细胞移植后,局部微环境中的炎症细胞释放TGF-β1,诱导肌源性干细胞成纤维分化,而影响了MDSCs成肌功能。探讨TGF-β1及其信号转导途径与肌源性干细胞成纤维分化之间的联系,对更深入的认识细胞移植治疗在体分化状况,提高细胞移植治疗效果具有重要意义。
本研究以SD大鼠为研究对象,利用酶消化法和密度梯度离心法相结合,分离纯化了MDSCs。利用RNA干扰技术,构建了靶向Smad3基因的慢病毒载体。体外shRNA抑制Smad3表达前后,Real-Time PCR及Western blot检测TGF-β1诱导的成纤维化/成肌分化标志蛋白表达变化;建立SUI大鼠模型,在体移植Smad3-shRNA基因修饰的MDSCs,检测抑制Smad3表达对大鼠尿控功能及MDSCs体内分化情况的影响。主要实验方法及结果如下:
一、MDSCs分离培养及成纤维分化的研究
1.通过胶原酶XI和Dispase酶联合消化分离和preplate差速贴壁技术,可以获得纯度较高的SD大鼠MDSCs,在适宜的培养条件MDSCs增殖活力较好,可稳定传代培养。
2. MDSCs的特征为:①低粘附性、小圆形、折光性强;②免疫表型:Sca-1(+)、CD34(+)、CD45(-)、Desmin(+);③有形成肌管的能力,但较肌卫星细胞弱。利用MDSCs的特征组成的鉴定系统可以作为鉴定MDSCs的可靠方法。
3.MDSCs能在TGF-β1刺激下表达成纤维分化标志蛋白Vimentin,而其成肌分化标志蛋白Desmin则被抑制,且存在明显的时间依赖关系,提示TGF-β1可能通过对MDSCs,即干细胞水平影响,参与了肌损伤修复/纤维化进程。
二、靶向Smad3基因RNA干扰慢病毒载体的构建与鉴定
1.设计4对siRNA干扰序列,并将其成功连接入pGCSIL-GFP载体,体外实验成功筛选出siRNA的最好靶点,靶点序列为:GGATGAAGTGTGTGTAAAT。
2.成功构建Smad3基因ShRNA慢病毒载体。用其感染MDSCs,感染效率在80%以上,此时Smad3蛋白表达的抑制效率达80%。
三、靶向干扰Smad3对MDSCs定向分化及SUI大鼠尿控功能的影响
1.Smad3基因RNA干扰慢病毒载体转染MDSCs可有效阻断TGF-β1诱导的Vimentin表达升高,Desmin表达下调。说明TGF-β1可能是通过激活Smad3蛋白表达,参与了肌损伤--纤维化的进程,阻断Smad3可能具有抑制MDSCs成纤维分化,维持其成肌特性的作用。
2.MDSCs细胞在体移植实验表明:MDSCs移植可修复替代损伤的括约肌细胞,能显著提高腹压漏尿点压(ALPP),改善尿失禁大鼠的控尿功能。抑制Smad3表达后,MDSCs成肌分化能力增强,显著提高SUI大鼠细胞移植治疗的远期疗效。
总之,本研究通过RNA干扰技术抑制MDSCs中Smad3基因表达,siRNA在有效抑制靶基因表达的同时可以有效降低成纤维化标志蛋白表达,维持成肌分化标志蛋白表达,改善尿失禁大鼠的控尿功能。本研究为更深入探讨TGF-β1及其信号转导途径与MDSCs成纤维化分化之间的联系,了解移植细胞在体分化状况,提高细胞移植治疗效果提供了新的线索。
Intrinsic sphincter deficiency(ISD) which is characterized by malfunction of the urethral closure is an important cause of stress urinary incontinence(SUI), especially in old women. There are still no long-term effective treatment for SUI due to ISD. Since patients with ISD have myogenic-dominant damages with urethral sphincter, stem cell transplantation may be a better way for repairing muscle injury so as to correct the defects in structure and function of sphincter urethral muscle thoroughly.
Muscle derived-stem cell(MDSCs),a highly undifferentiated multipotential cell,is characterized by rapid proliferation, slow fusing and multipotential differentiation. Its survival rate is significantly higher than that of satellite cells after transplantation. So it may be an ideal cell used for therapy of SUI with better alloimmunity regulation ability. Previous study showed that MDSCs injected into bladder smooth muscle layer and urethra survived and formed myofibrils, but Kwon compared MDSCs and fibroblasts in regard to their potential for restoration of urethral function following injection. The result revealed that no significant difference between MDSCs and fibroblasts or a combination of both, when the cell dosage was equal across the groups. The question whether the treatment effect of the MDSCs is hindered by the transdifferentiation of fibroblast is unknown.
The muscle recovery involves the competition between fibrosis and regeneration, therefore earlier stage recovery of traumatic muscles should be emphasized since more longer fibrosis would lead to less recovery possibility. under normal condition ,that source for the muscular recovery is derived from myogenic precursor cells and activated early in the healing process either by fusing with the local myofibers or by generating new myofibers. but the functional recovery of the muscle often is hindered by the transdifferentiation of fibroblast
Several studies have shown that myogenesis is also regulated by a number of growth factors, including fibroblast growth factor, insulin-like growth factor and transforming growth factor-b (TGF-b) TGF-βplays a key role in tissue repair and fibrosis, partly due to its capacity to induce myofibroblast differentiation. Smad3 is central in transducing TGF-b signaling during myogenesis.Smad3 mediates TGF-βsignaling related lung fibrosis and bronchial smooth muscle fibrosis.An in vivo study shows that loss of Smad3 greatly attenuated morphologic evidence of fibrosis in bleomycin-treated mice, thus implicating Smad3 in the pathogenesis of fibrosis. However, the Smad pathway and its possible role in mediating TGF-β1 induced MDSCs fibroblast differentiation have not been determined.
In this study we characterized the muscle-derived stem cells (MDSCs) isolated from rat gastrocnemius muscle, and examined whether blocking TGF-β1/Smad3 signaling pathway would inhibition the fibroblast differentiation effect of MDSCs in SUI rat.
Methods
The study was carried out from September 2006 to march 2009 at the department of Obstetrics and Gynecology, First Affiliated Hospital, Third Military Medical University. The experimental protocol was approved by Ethical committee for animal studies of the Third Military Medical University.
1 Materials
1.1 Experimental Animals
The experiments were performed on female Sprague-Dawley rats(3-4 weeks old) purchased from Experimental Animal Center, Research Institute of Third Military Medical University.
1.2 Isolation, culture and purification of MDSCs
The hind limbs (gastrocnemius muscles) were removed from 3~6 week-old S-D rats.Skin and bone were removed. The muscle mass was minced and chopped with razor blades, and the cells were dissociated using two enzymes (collagenase XI and dispase) for 1 h at 37℃.The muscle cell extract was preplated on culture flasks as described by Qu. The phenotypic characteristics of pp6 cells were detected by immunostaining for Sca-1, CD34 and desmin.
1.3 Immunocytochemical staining for muscle-derived stem cells
Cells were plated in a 6-well culture dish and fixed with cold methanol (-20℃) for 1 min. After rinsed with PBS, cells were blocked with 5% horse serum at room temperature for 30 min. The primary antibodies Desmin(1:200; Sigma), goat Sca-1(1:200 R&D),Vimentin(1:200 Sigmia))were applied overnight at room temperature. The immunofluorescence labeled second antibody(FITC-conjugated secondary antibodies or TRITC-conjugated secondary antibodies) were applied for 60 minutes at 37℃.Fluorescein isothiocyanate(FITC)-conjugated anti-CD34 antibodies was used to stain MDSCs. Nuclei were stained with DAPI(Beyotime). The cells were then rinsed with PBS and visualized by fluoroscopy.
1.4 Generation of lentiviral vector for silencing of rat Smad3 expression
From the Smad3 cDNA coding sequence: four pairs of cDNA oligonucleotides targeting Rat Smad3 mRNA at different locations were choosen (Table 1).These primers were annealed and inserted into pGCSIL-GFP (Genechem,Inc., Shanghai, China). Different siRNAs were screened by cotransfection with a rat Smad3 cDNA plasmid into 293T cells with Lipofectamine 2000 (Invitrogen Corporation, Carlsbad,CA, USA). To examine the efficiency of these targets in silencing Smad3 expression, Smad3 protein levels were measured using western blotting
The optimal sequence of siRNA against rat Smad3 was then cloned into the plasmid pGCL–GFP, which encodes an HIV-derived lentiviral vector containing a multiple cloning site for insertion of shRNA constructs to be driven by an upstream U6 promoter and a downstream cytomegalovirus promoter–GFP fluorescent protein cassette flanked by loxP sites. Lentivirus preparations were produced by the Shanghai GeneChem, Co. Ltd, China. The resulting lentiviral vector containing rat Smad3 shRNA was named GC-shSmad3, and its sequence was confirmed by PCR and sequencing analysis. MDSCs were infected with GC-shSmad3 by addition of lentivirus into the cell culture at an MOI of approximately 100. After 5 days of infection, MDSCs were serum starved for 24 h and then treated.
1.5 RNA isolation and qRT-PCR
Total RNA from MDSCs was extracted using Trizol reagent, and first-strand cDNA was generated using the ImProm-II? Reverse Transcription System (Promega). qRT-PCR was performed using the primers of Smad3 (5’-ACACAATAACTTGGACCTACAG-3’; 5’- GTGAAGCGTGGAATGTCTC-3). Vimentin(5’-TCCCTGAACCTGAGAGAAAC-3’; 5’- ATCGTGGTGCTG- AGAAGTC-3) Desmin(5’- CCTACACCTGCGAGATTGATG -3’; 5’- GCGATGTTGTCCTGATAGCC -3) Amplifications were performed in 45 cycles using an opticon continuous fluorescence detection system (MJ Research) with SYBR green fluorescence. Each cycle consisted of 5 s at 95℃, 30 s at 60℃. All samples were quantified using the comparative CT method for relative quantification of gene expression, normalized to GAPDH
1.6 Western blotting
Two hundred thousand MDSCs or MDSCs infected with GC-shSmad3 were seeded into 25-cm2 flasks in Dulbecco’s modified Eagle’s medium containing 10 ng/ml of TGF-_1 for 0、3、6、12、and 24 h. then the cells were lysed in buffer and the Iysates were separated by 10% SDS–PAGE. The proteins were transferred onto a nitrocellulose membrane (Bio-Rad). After incubation with mouse monoclonal anti-Vimentin (1:1 000 dilution; sigma), mouse monoclonal anti-Desmin (1:1 000 dilution; sigma)or Rabbit anti-smad3(1:200 dilution; Cellsignaling).The blot was washed with PBS and incubated with goat anti-mouse IgG conjugated with horseradish peroxidase (HRP; Zhongshan Biotech, China). Immunoreactive complexes were visualized by ECL and exposed to X-ray film. The density of Vimentin and Desmin protein bands was assayed by gel documentation system and QUANTITY ONE software.
1.7 Establishing rat model of SUI
The rats were given pentobarbital sodium anesthesia, surgery was performed through a dorsal incision in the skin and an incision in the muscle over the ischiorectal fossa. The pudendal nerve were exposed on each side and a 2 cm segment was removed just distal to the origin of the pudendal nerve from the sciatic nerve.
1.8 MDSCs Injection
female SD rats (250?300 g) were divided into 4 groups:Control (CON),denervated (SUI), denervated + MDSCs infected with a negative control lentiviral vector(MOCK),and denervated+ MDSCs infected with GC-shSmad3 (KD), The rats were given pentobarbital sodium anesthesia, a low midline incision was made to expose the bladder and urethra 50ul of cells suspension was injected with a microinjector, about 1×106 MSDCs per rat.
1.9 Urodynamic Test
2 weeks and 4 weeks after injection, urodynamic tests were performed.5 rats were taken from each group The rats were mounted on a table and placed in the dorsal position. A transvesical epidural catheter was inserted into the dome of the bladder, the other end of epidural catheter was connected with urodynamic detection and microinfusion pump through a three-way stopcock. Bladder emptied and saline solution was injected into bladder with the speed of 0.3ml/min.The pressure at the first drop leaked out from the urethral orifice was taken as leak point pressure(LPP).
1.10 Statistical analysis
Values are shown as means±SD. One-way ANOVA was used to compare the means from two or more experimental groups, followed by t-tests. Statistical differences between groups were considered to be significant at p < 0.05.
2 Results
2.1 Isolation and identification of MDSCs We isolated MDSCs from rat gastrocnemius muscles. The results show that immunofluorescence stains in muscle-derived cells are Desmin(+),CD34(+),Sca-1(+),CD45(-). They were therefore muscle-derived stem cells and were used for further experiments.
2.2 Suppress rat Smad3 expression by GC-shSmad3
Four shRNA-expressing plasmids were constructed using the pGCSIL-GFP vector to target Rat Smad3. One negative control shRNA containing a scrambled sequence with the same nucleotide composition was also constructed. The efficacy of these plasmids was examined by western blotting.
ShRNA plasmids showed variable efficacy, the most effective shRNA is shRNA1. The optimal sequence of siRNA against rat Smad3 (5’-GGATGAAGTGTGTGTAA AT-3’) was then cloned into the plasmid pGCL–GFP. MDSCs were infected with GC-shSmad3.The transfection efficiency was more than 80% showed by fluorescent microscopy analysis in MDSCs. The expression of Smad3 were monitored by quantitative realtime RT-PCR (qRT-PCR) and western blotting after the MDSCs cultured with TGF-β1 (10 ng/ml ) for 24 hours. With the most effective shRNA,shRNA1, The effective silencing of endogenous rat Smad3 by GC-shSmad3 was confirmed by western blot analysis, The Smad3 silencing rate was 80% confirmed by qRT-PCR
2.3 TGF-β1/Smad3 signaling in the fibroblast differentiation of muscle-derived stem cell
We have tested whether the stimulation of MDSCs cells with TGF-β1 will induce the expression of fibroblastic markers vimentin and whether blocking the TGF-β1/Smad3 signaling by vector-mediated Smad3 shRNA will suppress the fibroblast differentiation of muscle-derived stem cell. We also observed the changes of muscle-specific marker Desmin in these process. The result confirm that the expression of vimentin in the MDSCs can be induced by TGF-β1 stimulation in a time-dependent manner, but the expression of desmin were suppressed by TGF-β1.Inhibition of Smad3 with vector-mediated Smad3 shRNA significantly suppressed TGF-β1-induced effect of fibroblast differentiation.
2.4 Urodynamic Test after cell transplantation
Urinary dynamics detection for SUI models showed that LPP increased significantly. The phenomenon of SUI become weaken or disappear after injection. LPP in the MDSCs groups were significantly lower at 4 week than at 2 weeks. There were no such differences between 2 and 4 weeks in the CON and KD groups. LPP in the KD group were significantly higher than MOCK group at 4 week
3 Discussion
Taken together, We have isolated and purified MDSCs by the preplate technique. We Construct GC-shSmad3 lentiviral vector for silencing of rat Smad3 expression. The increased expressions of Vimentin and down-regulated of Desmin were found in MDSCs after culture with TGF-β1 in vitro, but TGF-β1 was unable to induce fibroblast differentiation of MDSCs in Smad3-deficient MDSCs, which exhibited accelerated myogenesis. In vivo testing showed that GC-shSmad3 lentivirus infection improved the MDSCs-mediated repair of urethral muscle, compared with MDSCs alone. Thus, we have shown GC-shSmad3 shRNA Inhibit the fibroblast differentiation of muscle-derived stem cell induced by TGF-β1 in vitro and improved the persistence of urethral muscle repair by preventing of muscle fibrosis in SUI model rat in vivo.
Myoblasts is a promising source for cell transplantation because they can easily be harvested from skeletal muscle and expanded in culture. Previous study has demonstrated a population of skeletal muscle-derived stem cells (MDSCs), characterized utilizing a variety of stem cell markers (e.g., Sca-1, CD-34, Bcl2) .These cells readily undergo differentiation into myotubes, which foster long-term persistence of the grafts formed within injected skeletal. It have been shown to be able of circumventing the limitations of myoblasts and could prove to be a superior alternative to myoblasts for the regeneration and repair of skeletal, cardiac and smooth muscles. But Several methods have been used to isolate and purify skeletal muscle-derived stem cell populations. The most popular method is established by Qu, They used three step enzyme(collagenase XI ,dispase and trypsin) digestion and the preplate technique to enrich the desired cells This method was obviously too tedious and uncertain for routine use. We explore a better way of isolating and purifying skeletal muscle stem cells by two enzyme(collagenase XI and dipase) digestion and the preplate technique and identified the MDSCs with immunocytochemistry. We have demonstrated two enzyme digestion is a better cell isolation protocol which can ease the procedure, decrease cell injury and improve cell quantity and quality.
The vectors have a high efficiency in gene delivery and can integrate genes into dividing stem cells,so we chose lentiviral vectors for gene transduction. Other virus vectors, such as adenovirus vectors, do not integrate into cells and thus the therapeutic transgene will be diluted over time. This may limit the use of adenoviral vectors for long-term expression of a potentially therapeutic gene. Our results show that high gene transduction efficiency (>80%) was achieved at day 5 after exposure to lentiviral vectors, suggesting gene integration.
TGF-β1 has been considered a key factor in the development of fibrosis in various tissue。It has been suggested that TGF-β1 is capable of inducing the expression of myofibroblastic markers in MC13 cells,This phenomenon is also demonstrated in our experiment, In addition, we have further observed the expression of Desmin was dramatically reduced upon TGF-β1.These data suggest that TGF-β1 is probably involved in the differentiation of MDSCs toward fibroblast, which is accompany with inhibition of myogenesis.
Smad3 is central in transducing TGF-β1 signaling during fibrosis and myogenesis. Therefore, we hypothesized that fibroblast transdifferentiation of MDSCs by TGF-β1 is also mediated by Smad3. We compared expression of Vimentin and Desmin in MDSCs infected with GC-shSmad3 lentivirus vector and MDSCs infected with a negative control lentiviral vector. The results have demonstrated that GC-shSmad3 lentivirus vector can attenuate the fibrosis reaction and this was concomitant with increased expression of differentiation-specific myogenic genes such as Desmin. The persistence of urethral muscle repair by preventing of muscle fibrosis was also observed by transplantation of MDSCs infected with GC-shSmad3 lentivirus vector to SUI model rat.
Although we cannot exclude the participation of other growth factors in the fibroblast transdifferentation of MDSCs, it seems possible based on these results that TGF-β1, the key factor involved in the fibrosis of various tissues, also plays a role in the differentation of fibroblast formation of MDSCs, which likely also contributes to the development of fibrosis. Furthermore, inhibition of Smad3 expression is capable of suppressing fibroblast differentiation of muscle-derived stem cell and improved the persistence of urethral muscle repair by preventing of muscle fibrosis in SUI model rat in vivo. It may provide a novel strategy for the treatment of SUI ,which may improve the effect of MDSCs treatment for female SUI patients.
导致SUI的关键机制主要有两种,尿道固有括约肌缺陷(intrinsic sphincter deficiency,ISD)和膀胱颈支撑功能障碍。ISD是导致女性SUI的重要原因,传统的治疗方法对于ISD所致的SUI的疗效差。从本质上改善ISD的方法是纠正其成肌功能障碍,即恢复ISD所致SUI患者成肌细胞功能。随着组织工程和基因治疗发展及干细胞研究深入,干细胞移植成为肌肉组织损伤修复的重要手段,这也为ISD型压力性尿失禁的治疗带来了希望。
肌源性干细胞(Muscle derived stem cells,MDSCs)是高度未分化的多潜能细胞,在适当条件下,MDSCs可分化为骨骼肌成肌细胞、造血细胞、成骨细胞、成软骨样细胞。研究发现骨骼肌损伤时,骨骼肌干细胞可分化形成肌纤维参与肌损伤修复过程。将MDSCs注入心肌梗死或尿失禁模型大鼠,MDSCs可参与心肌或尿道平滑肌的修复。在人体,已有成功用MDSCs治疗心肌梗死或压力性尿失禁的的研究报道。
目前MDSCs向具体细胞分化的机制尚不完全明确,可能与移植后细胞所处环境中的某些信号有关,不同的微环境可诱导MDSCs向不同的细胞分化,即MDSCs具有组织分化特异性。但是在MDSCs移植治疗骨骼肌损伤的研究中发现,MDSCs除分化为骨骼肌细胞外,在TGF-β等细胞因子的刺激下还可分化为成纤维细胞,导致骨骼肌纤维化,而影响细胞移植的疗效。在MDSCs移植治疗压力性尿失禁的研究中也发现:MDSCs或成纤维细胞移植到中段尿道周围,对尿失禁的治疗效果是相同的,提示:MDSCs移植后可能转分化为成纤维细胞参与尿道修复。
我们推测:肌源性干细胞移植后,局部微环境中的炎症细胞释放TGF-β1,诱导肌源性干细胞成纤维分化,而影响了MDSCs成肌功能。探讨TGF-β1及其信号转导途径与肌源性干细胞成纤维分化之间的联系,对更深入的认识细胞移植治疗在体分化状况,提高细胞移植治疗效果具有重要意义。
本研究以SD大鼠为研究对象,利用酶消化法和密度梯度离心法相结合,分离纯化了MDSCs。利用RNA干扰技术,构建了靶向Smad3基因的慢病毒载体。体外shRNA抑制Smad3表达前后,Real-Time PCR及Western blot检测TGF-β1诱导的成纤维化/成肌分化标志蛋白表达变化;建立SUI大鼠模型,在体移植Smad3-shRNA基因修饰的MDSCs,检测抑制Smad3表达对大鼠尿控功能及MDSCs体内分化情况的影响。主要实验方法及结果如下:
一、MDSCs分离培养及成纤维分化的研究
1.通过胶原酶XI和Dispase酶联合消化分离和preplate差速贴壁技术,可以获得纯度较高的SD大鼠MDSCs,在适宜的培养条件MDSCs增殖活力较好,可稳定传代培养。
2. MDSCs的特征为:①低粘附性、小圆形、折光性强;②免疫表型:Sca-1(+)、CD34(+)、CD45(-)、Desmin(+);③有形成肌管的能力,但较肌卫星细胞弱。利用MDSCs的特征组成的鉴定系统可以作为鉴定MDSCs的可靠方法。
3.MDSCs能在TGF-β1刺激下表达成纤维分化标志蛋白Vimentin,而其成肌分化标志蛋白Desmin则被抑制,且存在明显的时间依赖关系,提示TGF-β1可能通过对MDSCs,即干细胞水平影响,参与了肌损伤修复/纤维化进程。
二、靶向Smad3基因RNA干扰慢病毒载体的构建与鉴定
1.设计4对siRNA干扰序列,并将其成功连接入pGCSIL-GFP载体,体外实验成功筛选出siRNA的最好靶点,靶点序列为:GGATGAAGTGTGTGTAAAT。
2.成功构建Smad3基因ShRNA慢病毒载体。用其感染MDSCs,感染效率在80%以上,此时Smad3蛋白表达的抑制效率达80%。
三、靶向干扰Smad3对MDSCs定向分化及SUI大鼠尿控功能的影响
1.Smad3基因RNA干扰慢病毒载体转染MDSCs可有效阻断TGF-β1诱导的Vimentin表达升高,Desmin表达下调。说明TGF-β1可能是通过激活Smad3蛋白表达,参与了肌损伤--纤维化的进程,阻断Smad3可能具有抑制MDSCs成纤维分化,维持其成肌特性的作用。
2.MDSCs细胞在体移植实验表明:MDSCs移植可修复替代损伤的括约肌细胞,能显著提高腹压漏尿点压(ALPP),改善尿失禁大鼠的控尿功能。抑制Smad3表达后,MDSCs成肌分化能力增强,显著提高SUI大鼠细胞移植治疗的远期疗效。
总之,本研究通过RNA干扰技术抑制MDSCs中Smad3基因表达,siRNA在有效抑制靶基因表达的同时可以有效降低成纤维化标志蛋白表达,维持成肌分化标志蛋白表达,改善尿失禁大鼠的控尿功能。本研究为更深入探讨TGF-β1及其信号转导途径与MDSCs成纤维化分化之间的联系,了解移植细胞在体分化状况,提高细胞移植治疗效果提供了新的线索。
Intrinsic sphincter deficiency(ISD) which is characterized by malfunction of the urethral closure is an important cause of stress urinary incontinence(SUI), especially in old women. There are still no long-term effective treatment for SUI due to ISD. Since patients with ISD have myogenic-dominant damages with urethral sphincter, stem cell transplantation may be a better way for repairing muscle injury so as to correct the defects in structure and function of sphincter urethral muscle thoroughly.
Muscle derived-stem cell(MDSCs),a highly undifferentiated multipotential cell,is characterized by rapid proliferation, slow fusing and multipotential differentiation. Its survival rate is significantly higher than that of satellite cells after transplantation. So it may be an ideal cell used for therapy of SUI with better alloimmunity regulation ability. Previous study showed that MDSCs injected into bladder smooth muscle layer and urethra survived and formed myofibrils, but Kwon compared MDSCs and fibroblasts in regard to their potential for restoration of urethral function following injection. The result revealed that no significant difference between MDSCs and fibroblasts or a combination of both, when the cell dosage was equal across the groups. The question whether the treatment effect of the MDSCs is hindered by the transdifferentiation of fibroblast is unknown.
The muscle recovery involves the competition between fibrosis and regeneration, therefore earlier stage recovery of traumatic muscles should be emphasized since more longer fibrosis would lead to less recovery possibility. under normal condition ,that source for the muscular recovery is derived from myogenic precursor cells and activated early in the healing process either by fusing with the local myofibers or by generating new myofibers. but the functional recovery of the muscle often is hindered by the transdifferentiation of fibroblast
Several studies have shown that myogenesis is also regulated by a number of growth factors, including fibroblast growth factor, insulin-like growth factor and transforming growth factor-b (TGF-b) TGF-βplays a key role in tissue repair and fibrosis, partly due to its capacity to induce myofibroblast differentiation. Smad3 is central in transducing TGF-b signaling during myogenesis.Smad3 mediates TGF-βsignaling related lung fibrosis and bronchial smooth muscle fibrosis.An in vivo study shows that loss of Smad3 greatly attenuated morphologic evidence of fibrosis in bleomycin-treated mice, thus implicating Smad3 in the pathogenesis of fibrosis. However, the Smad pathway and its possible role in mediating TGF-β1 induced MDSCs fibroblast differentiation have not been determined.
In this study we characterized the muscle-derived stem cells (MDSCs) isolated from rat gastrocnemius muscle, and examined whether blocking TGF-β1/Smad3 signaling pathway would inhibition the fibroblast differentiation effect of MDSCs in SUI rat.
Methods
The study was carried out from September 2006 to march 2009 at the department of Obstetrics and Gynecology, First Affiliated Hospital, Third Military Medical University. The experimental protocol was approved by Ethical committee for animal studies of the Third Military Medical University.
1 Materials
1.1 Experimental Animals
The experiments were performed on female Sprague-Dawley rats(3-4 weeks old) purchased from Experimental Animal Center, Research Institute of Third Military Medical University.
1.2 Isolation, culture and purification of MDSCs
The hind limbs (gastrocnemius muscles) were removed from 3~6 week-old S-D rats.Skin and bone were removed. The muscle mass was minced and chopped with razor blades, and the cells were dissociated using two enzymes (collagenase XI and dispase) for 1 h at 37℃.The muscle cell extract was preplated on culture flasks as described by Qu. The phenotypic characteristics of pp6 cells were detected by immunostaining for Sca-1, CD34 and desmin.
1.3 Immunocytochemical staining for muscle-derived stem cells
Cells were plated in a 6-well culture dish and fixed with cold methanol (-20℃) for 1 min. After rinsed with PBS, cells were blocked with 5% horse serum at room temperature for 30 min. The primary antibodies Desmin(1:200; Sigma), goat Sca-1(1:200 R&D),Vimentin(1:200 Sigmia))were applied overnight at room temperature. The immunofluorescence labeled second antibody(FITC-conjugated secondary antibodies or TRITC-conjugated secondary antibodies) were applied for 60 minutes at 37℃.Fluorescein isothiocyanate(FITC)-conjugated anti-CD34 antibodies was used to stain MDSCs. Nuclei were stained with DAPI(Beyotime). The cells were then rinsed with PBS and visualized by fluoroscopy.
1.4 Generation of lentiviral vector for silencing of rat Smad3 expression
From the Smad3 cDNA coding sequence: four pairs of cDNA oligonucleotides targeting Rat Smad3 mRNA at different locations were choosen (Table 1).These primers were annealed and inserted into pGCSIL-GFP (Genechem,Inc., Shanghai, China). Different siRNAs were screened by cotransfection with a rat Smad3 cDNA plasmid into 293T cells with Lipofectamine 2000 (Invitrogen Corporation, Carlsbad,CA, USA). To examine the efficiency of these targets in silencing Smad3 expression, Smad3 protein levels were measured using western blotting
The optimal sequence of siRNA against rat Smad3 was then cloned into the plasmid pGCL–GFP, which encodes an HIV-derived lentiviral vector containing a multiple cloning site for insertion of shRNA constructs to be driven by an upstream U6 promoter and a downstream cytomegalovirus promoter–GFP fluorescent protein cassette flanked by loxP sites. Lentivirus preparations were produced by the Shanghai GeneChem, Co. Ltd, China. The resulting lentiviral vector containing rat Smad3 shRNA was named GC-shSmad3, and its sequence was confirmed by PCR and sequencing analysis. MDSCs were infected with GC-shSmad3 by addition of lentivirus into the cell culture at an MOI of approximately 100. After 5 days of infection, MDSCs were serum starved for 24 h and then treated.
1.5 RNA isolation and qRT-PCR
Total RNA from MDSCs was extracted using Trizol reagent, and first-strand cDNA was generated using the ImProm-II? Reverse Transcription System (Promega). qRT-PCR was performed using the primers of Smad3 (5’-ACACAATAACTTGGACCTACAG-3’; 5’- GTGAAGCGTGGAATGTCTC-3). Vimentin(5’-TCCCTGAACCTGAGAGAAAC-3’; 5’- ATCGTGGTGCTG- AGAAGTC-3) Desmin(5’- CCTACACCTGCGAGATTGATG -3’; 5’- GCGATGTTGTCCTGATAGCC -3) Amplifications were performed in 45 cycles using an opticon continuous fluorescence detection system (MJ Research) with SYBR green fluorescence. Each cycle consisted of 5 s at 95℃, 30 s at 60℃. All samples were quantified using the comparative CT method for relative quantification of gene expression, normalized to GAPDH
1.6 Western blotting
Two hundred thousand MDSCs or MDSCs infected with GC-shSmad3 were seeded into 25-cm2 flasks in Dulbecco’s modified Eagle’s medium containing 10 ng/ml of TGF-_1 for 0、3、6、12、and 24 h. then the cells were lysed in buffer and the Iysates were separated by 10% SDS–PAGE. The proteins were transferred onto a nitrocellulose membrane (Bio-Rad). After incubation with mouse monoclonal anti-Vimentin (1:1 000 dilution; sigma), mouse monoclonal anti-Desmin (1:1 000 dilution; sigma)or Rabbit anti-smad3(1:200 dilution; Cellsignaling).The blot was washed with PBS and incubated with goat anti-mouse IgG conjugated with horseradish peroxidase (HRP; Zhongshan Biotech, China). Immunoreactive complexes were visualized by ECL and exposed to X-ray film. The density of Vimentin and Desmin protein bands was assayed by gel documentation system and QUANTITY ONE software.
1.7 Establishing rat model of SUI
The rats were given pentobarbital sodium anesthesia, surgery was performed through a dorsal incision in the skin and an incision in the muscle over the ischiorectal fossa. The pudendal nerve were exposed on each side and a 2 cm segment was removed just distal to the origin of the pudendal nerve from the sciatic nerve.
1.8 MDSCs Injection
female SD rats (250?300 g) were divided into 4 groups:Control (CON),denervated (SUI), denervated + MDSCs infected with a negative control lentiviral vector(MOCK),and denervated+ MDSCs infected with GC-shSmad3 (KD), The rats were given pentobarbital sodium anesthesia, a low midline incision was made to expose the bladder and urethra 50ul of cells suspension was injected with a microinjector, about 1×106 MSDCs per rat.
1.9 Urodynamic Test
2 weeks and 4 weeks after injection, urodynamic tests were performed.5 rats were taken from each group The rats were mounted on a table and placed in the dorsal position. A transvesical epidural catheter was inserted into the dome of the bladder, the other end of epidural catheter was connected with urodynamic detection and microinfusion pump through a three-way stopcock. Bladder emptied and saline solution was injected into bladder with the speed of 0.3ml/min.The pressure at the first drop leaked out from the urethral orifice was taken as leak point pressure(LPP).
1.10 Statistical analysis
Values are shown as means±SD. One-way ANOVA was used to compare the means from two or more experimental groups, followed by t-tests. Statistical differences between groups were considered to be significant at p < 0.05.
2 Results
2.1 Isolation and identification of MDSCs We isolated MDSCs from rat gastrocnemius muscles. The results show that immunofluorescence stains in muscle-derived cells are Desmin(+),CD34(+),Sca-1(+),CD45(-). They were therefore muscle-derived stem cells and were used for further experiments.
2.2 Suppress rat Smad3 expression by GC-shSmad3
Four shRNA-expressing plasmids were constructed using the pGCSIL-GFP vector to target Rat Smad3. One negative control shRNA containing a scrambled sequence with the same nucleotide composition was also constructed. The efficacy of these plasmids was examined by western blotting.
ShRNA plasmids showed variable efficacy, the most effective shRNA is shRNA1. The optimal sequence of siRNA against rat Smad3 (5’-GGATGAAGTGTGTGTAA AT-3’) was then cloned into the plasmid pGCL–GFP. MDSCs were infected with GC-shSmad3.The transfection efficiency was more than 80% showed by fluorescent microscopy analysis in MDSCs. The expression of Smad3 were monitored by quantitative realtime RT-PCR (qRT-PCR) and western blotting after the MDSCs cultured with TGF-β1 (10 ng/ml ) for 24 hours. With the most effective shRNA,shRNA1, The effective silencing of endogenous rat Smad3 by GC-shSmad3 was confirmed by western blot analysis, The Smad3 silencing rate was 80% confirmed by qRT-PCR
2.3 TGF-β1/Smad3 signaling in the fibroblast differentiation of muscle-derived stem cell
We have tested whether the stimulation of MDSCs cells with TGF-β1 will induce the expression of fibroblastic markers vimentin and whether blocking the TGF-β1/Smad3 signaling by vector-mediated Smad3 shRNA will suppress the fibroblast differentiation of muscle-derived stem cell. We also observed the changes of muscle-specific marker Desmin in these process. The result confirm that the expression of vimentin in the MDSCs can be induced by TGF-β1 stimulation in a time-dependent manner, but the expression of desmin were suppressed by TGF-β1.Inhibition of Smad3 with vector-mediated Smad3 shRNA significantly suppressed TGF-β1-induced effect of fibroblast differentiation.
2.4 Urodynamic Test after cell transplantation
Urinary dynamics detection for SUI models showed that LPP increased significantly. The phenomenon of SUI become weaken or disappear after injection. LPP in the MDSCs groups were significantly lower at 4 week than at 2 weeks. There were no such differences between 2 and 4 weeks in the CON and KD groups. LPP in the KD group were significantly higher than MOCK group at 4 week
3 Discussion
Taken together, We have isolated and purified MDSCs by the preplate technique. We Construct GC-shSmad3 lentiviral vector for silencing of rat Smad3 expression. The increased expressions of Vimentin and down-regulated of Desmin were found in MDSCs after culture with TGF-β1 in vitro, but TGF-β1 was unable to induce fibroblast differentiation of MDSCs in Smad3-deficient MDSCs, which exhibited accelerated myogenesis. In vivo testing showed that GC-shSmad3 lentivirus infection improved the MDSCs-mediated repair of urethral muscle, compared with MDSCs alone. Thus, we have shown GC-shSmad3 shRNA Inhibit the fibroblast differentiation of muscle-derived stem cell induced by TGF-β1 in vitro and improved the persistence of urethral muscle repair by preventing of muscle fibrosis in SUI model rat in vivo.
Myoblasts is a promising source for cell transplantation because they can easily be harvested from skeletal muscle and expanded in culture. Previous study has demonstrated a population of skeletal muscle-derived stem cells (MDSCs), characterized utilizing a variety of stem cell markers (e.g., Sca-1, CD-34, Bcl2) .These cells readily undergo differentiation into myotubes, which foster long-term persistence of the grafts formed within injected skeletal. It have been shown to be able of circumventing the limitations of myoblasts and could prove to be a superior alternative to myoblasts for the regeneration and repair of skeletal, cardiac and smooth muscles. But Several methods have been used to isolate and purify skeletal muscle-derived stem cell populations. The most popular method is established by Qu, They used three step enzyme(collagenase XI ,dispase and trypsin) digestion and the preplate technique to enrich the desired cells This method was obviously too tedious and uncertain for routine use. We explore a better way of isolating and purifying skeletal muscle stem cells by two enzyme(collagenase XI and dipase) digestion and the preplate technique and identified the MDSCs with immunocytochemistry. We have demonstrated two enzyme digestion is a better cell isolation protocol which can ease the procedure, decrease cell injury and improve cell quantity and quality.
The vectors have a high efficiency in gene delivery and can integrate genes into dividing stem cells,so we chose lentiviral vectors for gene transduction. Other virus vectors, such as adenovirus vectors, do not integrate into cells and thus the therapeutic transgene will be diluted over time. This may limit the use of adenoviral vectors for long-term expression of a potentially therapeutic gene. Our results show that high gene transduction efficiency (>80%) was achieved at day 5 after exposure to lentiviral vectors, suggesting gene integration.
TGF-β1 has been considered a key factor in the development of fibrosis in various tissue。It has been suggested that TGF-β1 is capable of inducing the expression of myofibroblastic markers in MC13 cells,This phenomenon is also demonstrated in our experiment, In addition, we have further observed the expression of Desmin was dramatically reduced upon TGF-β1.These data suggest that TGF-β1 is probably involved in the differentiation of MDSCs toward fibroblast, which is accompany with inhibition of myogenesis.
Smad3 is central in transducing TGF-β1 signaling during fibrosis and myogenesis. Therefore, we hypothesized that fibroblast transdifferentiation of MDSCs by TGF-β1 is also mediated by Smad3. We compared expression of Vimentin and Desmin in MDSCs infected with GC-shSmad3 lentivirus vector and MDSCs infected with a negative control lentiviral vector. The results have demonstrated that GC-shSmad3 lentivirus vector can attenuate the fibrosis reaction and this was concomitant with increased expression of differentiation-specific myogenic genes such as Desmin. The persistence of urethral muscle repair by preventing of muscle fibrosis was also observed by transplantation of MDSCs infected with GC-shSmad3 lentivirus vector to SUI model rat.
Although we cannot exclude the participation of other growth factors in the fibroblast transdifferentation of MDSCs, it seems possible based on these results that TGF-β1, the key factor involved in the fibrosis of various tissues, also plays a role in the differentation of fibroblast formation of MDSCs, which likely also contributes to the development of fibrosis. Furthermore, inhibition of Smad3 expression is capable of suppressing fibroblast differentiation of muscle-derived stem cell and improved the persistence of urethral muscle repair by preventing of muscle fibrosis in SUI model rat in vivo. It may provide a novel strategy for the treatment of SUI ,which may improve the effect of MDSCs treatment for female SUI patients.
引文
1. Abrams P, Cardozo L, Fall M, et al. The standardisation of terminology in lower urinary tract function: report from the standardisation sub-committee of the International Continence Society. Urology 2003;61(1):37-49.
2. Hunskaar S, Lose G, Sykes D, Voss S. The prevalence of urinary incontinence in women in four European countries. BJU Int 2004;93(3):324-30.
3. Hunskaar S, Burgio K, Diokno A, Herzog AR, Hjalmas K, Lapitan MC. Epidemiology and natural history of urinary incontinence in women. Urology 2003;62(4 Suppl 1):16-23.
4. Miller DC, Saigal CS, Litwin MS. The demographic burden of urologic diseases in America. Urol Clin North Am 2009;36(1):11-27, v.
5. NIH state-of-the-science conference statement on prevention of fecal and urinary incontinence in adults. NIH Consens State Sci Statements 2007;24(1):1-37.
6. Farage MA, Miller KW, Berardesca E, Maibach HI. Psychosocial and societal burden of incontinence in the aged population: a review. Arch Gynecol Obstet 2008;277(4):285-90.
7. Kevorkian R. Physiology of incontinence. Clin Geriatr Med 2004;20(3):409-25, v.
8. Takahashi S, Homma Y, Fujishiro T, Hosaka Y, Kitamura T, Kawabe K. Electromyographic study of the striated urethral sphincter in type 3 stress incontinence: evidence of myogenic-dominant damages. Urology 2000;56(6):946-50.
9. Hasiam J. Current treatments for patients with stress urinary incontinence. Nurs Times 2004;100(2):50-1.
10. Strasser H, Marksteiner R, Margreiter E, et al. [Stem cell therapy for urinary incontinence]. Urologe A 2004;43(10):1237-41.
11. Mitterberger M, Pinggera GM, Marksteiner R, et al. Adult stem cell therapy of female stress urinary incontinence. Eur Urol 2008;53(1):169-75.
12. Cao B, Huard J. Muscle-derived stem cells. Cell Cycle 2004;3(2):104-7.
13. Charge SB, Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev 2004;84(1):209-38.
14. Pagani FD, DerSimonian H, Zawadzka A, et al. Autologous skeletal myoblaststransplanted to ischemia-damaged myocardium in humans. Histological analysis of cell survival and differentiation. J Am Coll Cardiol 2003;41(5):879-88.
15. Herreros J, Prosper F, Perez A, et al. Autologous intramyocardial injection of cultured skeletal muscle-derived stem cells in patients with non-acute myocardial infarction. Eur Heart J 2003;24(22):2012-20.
16. Howell JC, Lee WH, Morrison P, Zhong J, Yoder MC, Srour EF. Pluripotent stem cells identified in multiple murine tissues. Ann N Y Acad Sci 2003;996:158-73.
17. Cannon TW, Lee JY, Somogyi G, Pruchnic R, Smith CP, Huard J, Chancellor MB. Improved sphincter contractility after allogenic muscle-derived progenitor cell injection into the denervated rat urethra. Urology 2003;62(5):958-63.
18. Lee JY, Paik SY, Yuk SH, Lee JH, Ghil SH, Lee SS. Long term effects of muscle-derived stem cells on leak point pressure and closing pressure in rats with transected pudendal nerves. Mol Cells 2004;18(3):309-13.
19. Chermansky CJ, Tarin T, Kwon DD, et al. Intraurethral muscle-derived cell injections increase leak point pressure in a rat model of intrinsic sphincter deficiency. Urology 2004;63(4):780-5.
20. Lee JY, Cannon TW, Pruchnic R, Fraser MO, Huard J, Chancellor MB. The effects of periurethral muscle-derived stem cell injection on leak point pressure in a rat model of stress urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct 2003;14(1):31-7; discussion 37.
21. Liu D, Black BL, Derynck R. TGF-βinhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3. Genes Dev 2001;15(22):2950-66.
22. Liu D, Kang JS, Derynck R. TGF-beta-activated Smad3 represses MEF2-dependent transcription in myogenic differentiation. EMBO J 2004;23(7):1557-66.
23. Li Y, Foster W, Deasy BM, et al. Transforming growth factor-beta1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis. Am J Pathol 2004;164(3):1007-19.
24. Kwon D, Kim Y, Pruchnic R, et al. Periurethral cellular injection: comparison of muscle-derived progenitor cells and fibroblasts with regard to efficacy and tissue contractility in an animal model of stress urinary incontinence. Urology 2006;68(2):449-54.
25. Chamberlain JR, Schwarze U, Wang PR, et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 2004;303(5661):1198-201.
26. Qu-Petersen Z, Deasy B, Jankowski R, et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol 2002;157(5):851-64.
27. Lee JY, Qu-Petersen Z, Cao B, et al. Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing. J Cell Biol 2000;150(5):1085-100.
28. Gharaibeh B, Lu A, Tebbets J, et al. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc 2008;3(9):1501-9.
29.姚启盛,叶章群,陈丛波,王小康,王卫民,陈立新. Dispase在成年鼠骨骼肌干细胞纯化中的价值.郧阳医学院学报; 2004:336-337.
30. Huard J, Cao B, Qu-Petersen Z. Muscle-derived stem cells: potential for muscle regeneration. Birth Defects Res C Embryo Today 2003;69(3):230-7.
31. Li Y, Huard J. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. Am J Pathol 2002;161(3):895-907.
32. Zheng B, Cao B, Crisan M, et al. Prospective identification of myogenic endothelial cells in human skeletal muscle. Nat Biotechnol 2007;25(9):1025-34.
33. Lu A, Cummins JH, Pollett JB, Cao B, Sun B, Rudnicki MA, Huard J. Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics. Gene Ther 2008;15(15):1116-25.
34. Jankowski RJ, Haluszczak C, Trucco M, Huard J. Flow cytometric characterization of myogenic cell populations obtained via the preplate technique: potential for rapid isolation of muscle-derived stem cells. Hum Gene Ther 2001;12(6):619-28.
35. Deasy BM, Lu A, Tebbets JC, et al. A role for cell sex in stem cell-mediated skeletal muscle regeneration: female cells have higher muscle regeneration efficiency. J Cell Biol 2007;177(1):73-86.
36. Roberts AB, Russo A, Felici A, Flanders KC. Smad3: a key player in pathogenetic mechanisms dependent on TGF-beta. Ann N Y Acad Sci 2003;995:1-10.
37. Flanders KC, Sullivan CD, Fujii M, et al. Mice lacking Smad3 are protected againstcutaneous injury induced by ionizing radiation. Am J Pathol 2002;160(3):1057-68.
38. Danisovic L, Varga I, Polak S, Ulicna M, Bohmer D, Vojtassak J. Morphology of in vitro expanded human muscle-derived stem cells. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2008;152(2):235-8.
39. Schnaper HW, Hayashida T, Hubchak SC, Poncelet AC. TGF-βsignal transduction and mesangial cell fibrogenesis. Am J Physiol Renal Physiol 2003;284(2):F243-52.
40. Verrecchia F, Mauviel A. Transforming growth factor-beta and fibrosis. World J Gastroenterol 2007;13(22):3056-62.
41. Verrecchia F, Laboureau J, Verola O, et al. Skin involvement in scleroderma--where histological and clinical scores meet. Rheumatology (Oxford) 2007;46(5):833-41.
42. Shi Y, Massague J. Mechanisms of TGF-βsignaling from cell membrane to the nucleus. Cell 2003;113(6):685-700.
43. Attisano L, Wrana JL. Smads as transcriptional co-modulators. Curr Opin Cell Biol 2000;12(2):235-43.
44. Chen W, Fu X, Sheng Z. Review of current progress in the structure and function of Smad proteins. Chin Med J (Engl) 2002;115(3):446-50.
45. Scherr M, Eder M. Gene transfer into hematopoietic stem cells using lentiviral vectors. Curr Gene Ther 2002;2(1):45-55.
46. Sato M, Markiewicz M, Yamanaka M, et al. Modulation of transforming growth factor-beta (TGF-beta) signaling by endogenous sphingolipid mediators. J Biol Chem 2003;278(11):9276-82.
47. Verrecchia F, Chu ML, Mauviel A. Identification of novel TGF-β/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach. J Biol Chem 2001;276(20):17058-62.
48. Zhao J, Shi W, Wang YL, et al. Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol 2002;282(3): L585-93.
49. Schnabl B, Kweon YO, Frederick JP, Wang XF, Rippe RA, Brenner DA. The role of Smad3 in mediating mouse hepatic stellate cell activation. Hepatology 2001;34(1):89-100.
50. Fujimoto M, Maezawa Y, Yokote K, et al. Mice lacking Smad3 are protected againststreptozotocin-induced diabetic glomerulopathy. Biochem Biophys Res Commun 2003;305(4):1002-7.
51. Scherr M, Battmer K, Blomer U, Schiedlmeier B, Ganser A, Grez M, Eder M. Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells. Blood 2002;99(2):709-12.
52. Lan HY, Mu W, Tomita N, et al. Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model. J Am Soc Nephrol 2003;14(6):1535-48.
53. Terada Y, Hanada S, Nakao A, Kuwahara M, Sasaki S, Marumo F. Gene transfer of Smad7 using electroporation of adenovirus prevents renal fibrosis in post-obstructed kidney. Kidney Int 2002;61(1 Suppl):S94-8.
54. Nakao A, Fujii M, Matsumura R, Kumano K, Saito Y, Miyazono K, Iwamoto I. Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice. J Clin Invest 1999;104(1):5-11.
55. Pines M, Snyder D, Yarkoni S, Nagler A. Halofuginone to treat fibrosis in chronic graft-versus-host disease and scleroderma. Biol Blood Marrow Transplant 2003;9(7):417-25.
56. Bruck R, Genina O, Aeed H, Alexiev R, Nagler A, Avni Y, Pines M. Halofuginone to prevent and treat thioacetamide-induced liver fibrosis in rats. Hepatology 2001;33(2):379-86.
57. McGaha TL, Phelps RG, Spiera H, Bona C. Halofuginone, an inhibitor of type-I collagen synthesis and skin sclerosis, blocks transforming-growth-factor-beta-mediated Smad3 activation in fibroblasts. J Invest Dermatol 2002;118(3):461-70.
58. Nishitsuji H, Ikeda T, Miyoshi H, Ohashi T, Kannagi M, Masuda T. Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary non-dividing cells. Microbes Infect 2004;6(1):76-85.
59. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296(5567):550-3.
60. Ono A, Utsugi M, Masubuchi K, et al. Glutathione redox regulates TGF-beta-induced fibrogenic effects through Smad3 activation. FEBS Lett 2009;583(2):357-62.
61. Holterman CE, Rudnicki MA. Molecular regulation of satellite cell function. Semin Cell Dev Biol 2005;16(4-5):575-84.
62. Cornelison DD, Filla MS, Stanley HM, Rapraeger AC, Olwin BB. Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration. Dev Biol 2001;239(1):79-94.
63.徐惠成,王延洲,等.肌源性干细胞治疗压力性尿失禁的实验研究.第三军医大学学报2007年02期; 2007:113-5.
64. Huard J, Yokoyama T, Pruchnic R, et al. Muscle-derived cell-mediated ex vivo gene therapy for urological dysfunction. Gene Ther 2002;9(23):1617-26.
65. Yokoyama T, Pruchnic R, Lee JY, et al. Autologous primary muscle-derived cells transfer into the lower urinary tract. Tissue Eng 2001;7(4):395-404.
66. Chancellor MB, Yokoyama T, Tirney S, et al. Preliminary results of myoblast injection into the urethra and bladder wall: a possible method for the treatment of stress urinary incontinence and impaired detrusor contractility. Neurourol Urodyn 2000;19(3): 279-87.
1. Hannestad YS, Rortveit G, Sandvik H, Hunskaar S. A community-based epidemiological survey of female urinary incontinence: the Norwegian EPINCONT study. Epidemiology of Incontinence in the County of Nord-Trondelag. J Clin Epidemiol 2000;53(11):1150-7.
2. Subak LL, Whitcomb E, Shen H, Saxton J, Vittinghoff E, Brown JS. Weight loss: a novel and effective treatment for urinary incontinence. J Urol 2005;174(1):190-5.
3. Swithinbank L, Hashim H, Abrams P. The effect of fluid intake on urinary symptoms in women. J Urol 2005;174(1):187-9.
4. Hannestad YS, Rortveit G, Daltveit AK, Hunskaar S. Are smoking and other lifestyle factors associated with female urinary incontinence? The Norwegian EPINCONT Study. BJOG 2003;110(3):247-54.
5. Bo K, Borgen JS. Prevalence of stress and urge urinary incontinence in elite athletes and controls. Med Sci Sports Exerc 2001;33(11):1797-802.
6. Richter HE, Burgio KL, Goode PS, et al. Non-surgical management of stress urinary incontinence: ambulatory treatments for leakage associated with stress (ATLAS) trial. Clin Trials 2007;4(1):92-101.
7. Hay-Smith EJ, Bo Berghmans LC, Hendriks HJ, de Bie RA, van Waalwijk van Doorn ES. Pelvic floor muscle training for urinary incontinence in women. Cochrane Database Syst Rev 2001(1):CD001407.
8. Dumoulin C, Hay-Smith J. Pelvic floor muscle training versus no treatment for urinary incontinence in women. A Cochrane systematic review. Eur J Phys Rehabil Med 2008;44(1):47-63.
9. Aksac B, Aki S, Karan A, Yalcin O, Isikoglu M, Eskiyurt N. Biofeedback and pelvic floor exercises for the rehabilitation of urinary stress incontinence. Gynecol ObstetInvest 2003;56(1):23-7.
10. Spruijt J, Vierhout M, Verstraeten R, Janssens J, Burger C. Vaginal electrical stimulation of the pelvic floor: a randomized feasibility study in urinary incontinent elderly women. Acta Obstet Gynecol Scand 2003;82(11):1043-8.
11. Alhasso A, Glazener CM, Pickard R, N'dow J. Adrenergic drugs for urinary incontinence in adults. Cochrane Database Syst Rev 2005(3):CD001842.
12. Boy S, Reitz A, Wirth B, Knapp PA, Braun PM, Haferkamp A, Schurch B. Facilitatory neuromodulative effect of duloxetine on pudendal motor neurons controlling the urethral pressure: a functional urodynamic study in healthy women. Eur Urol 2006;50(1):119-25.
13. Athanasiou S, Chaliha C, Digesu GA, Sotiropoulou M, Georgoulias N, Khullar V, Antsaklis A. The effects of duloxetine on urethral function and sphincter morphology. Int Urogynecol J Pelvic Floor Dysfunct 2007;18(7):763-7.
14. Lin AT, Sun MJ, Tai HL, et al. Duloxetine versus placebo for the treatment of women with stress predominant urinary incontinence in Taiwan: a double-blind, randomized, placebo-controlled trial. BMC Urol 2008;8:2.
15. Cardozo L, Drutz HP, Baygani SK, Bump RC. Pharmacological treatment of women awaiting surgery for stress urinary incontinence. Obstet Gynecol 2004;104(3):511-9.
16. Duckett J, Patil A, Aggarwal I. The effect of duloxetine on urethral sphincter morphology. Ultrasound Obstet Gynecol 2008;31(2):206-9.
17. Ghoniem GM, Van Leeuwen JS, Elser DM, Freeman RM, Zhao YD, Yalcin I, Bump RC. A randomized controlled trial of duloxetine alone, pelvic floor muscle training alone, combined treatment and no active treatment in women with stress urinary incontinence. J Urol 2005;173(5):1647-53.
18. Hurley DJ, Turner CL, Yalcin I, Viktrup L, Baygani SK. Duloxetine for the treatment of stress urinary incontinence in women: an integrated analysis of safety. Eur J Obstet Gynecol Reprod Biol 2006;125(1):120-8.
19. Mitterberger M, Pinggera GM, Marksteiner R, et al. Adult stem cell therapy of female stress urinary incontinence. Eur Urol 2008;53(1):169-75.
20. Duckett JR, Vella M, Kavalakuntla G, Basu M. Tolerability and efficacy of duloxetine in a nontrial situation. BJOG 2007;114(5):543-7.
21. Vella M, Duckett J, Basu M. Duloxetine 1 year on: the long-term outcome of a cohort of women prescribed duloxetine. Int Urogynecol J Pelvic Floor Dysfunct 2008;19(7):961-4.
22. Basu M, Duckett J. The treatment of urinary incontinence with Duloxetine. J Obstet Gynaecol 2008;28(2):166-9.
23. Grady D, Brown JS, Vittinghoff E, Applegate W, Varner E, Snyder T. Postmenopausal hormones and incontinence: the Heart and Estrogen/Progestin Replacement Study. Obstet Gynecol 2001;97(1):116-20.
24. Latthe PM, Foon R, Khan K. Nonsurgical treatment of stress urinary incontinence (SUI): grading of evidence in systematic reviews. BJOG 2008;115(4):435-44.
25. Castro-Diaz D, Amoros MA. Pharmacotherapy for stress urinary incontinence. Curr Opin Urol 2005;15(4):227-30.
26. Schagen van Leeuwen JH, Lange RR, Jonasson AF, Chen WJ, Viktrup L. Efficacy and safety of duloxetine in elderly women with stress urinary incontinence or stress-predominant mixed urinary incontinence. Maturitas 2008;60(2):138-47.
1. Corcos J, Beaulieu S, Donovan J, Naughton M, Gotoh M. Quality of life assessment in men and women with urinary incontinence. J Urol 2002;168(3):896-905.
2. Norton P, Brubaker L. Urinary incontinence in women. Lancet 2006;367(9504):57-67.
3. Meyer S, Schreyer A, De Grandi P, Hohlfeld P. The effects of birth on urinary continence mechanisms and other pelvic-floor characteristics. Obstet Gynecol 1998;92(4 Pt 1):613-8.
4. Radley SC, Chapple CR, Bryan NP, Clarke DE, Craig DA. Effect of methoxamine on maximum urethral pressure in women with genuine stress incontinence: a placebo- controlled, double-blind crossover study. Neurourol Urodyn 2001;20(1):43-52.
5. Viktrup L, Perahia DG, Tylee A. Duloxetine Treatment of Stress Urinary Incontinence in Women Does Not Induce Mania or Hypomania. Prim Care Companion J Clin Psychiatry 2004;6(6):239-243.
6. Dewan PA, Byard RW. Re: Transurethral Polytef injection for post-prostatectomy urinary incontinence and Long-term follow-up of women treated with periurethral Teflon injections for stress incontinence. Br J Urol 1993;71(1):112.
7. Bent AE, Tutrone RT, McLennan MT, Lloyd LK, Kennelly MJ, Badlani G. Treatment of intrinsic sphincter deficiency using autologous ear chondrocytes as a bulking agent. Neurourol Urodyn 2001;20(2):157-65.
8. Lee JY, Cannon TW, Pruchnic R, Fraser MO, Huard J, Chancellor MB. The effects of periurethral muscle-derived stem cell injection on leak point pressure in a rat model of stress urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct 2003;14(1):31-7; discussion 37.
9. DeLancey JO. Structural support of the urethra as it relates to stress urinary incontinence: the hammock hypothesis. Am J Obstet Gynecol 1994;170(6):1713-20; discussion 1720-3.
10. de Groat WC. Integrative control of the lower urinary tract: preclinical perspective. Br J Pharmacol 2006;147 Suppl 2:S25-40.
11. Kaiho Y, Kamo I, Chancellor MB, Arai Y, de Groat WC, Yoshimura N. Role of noradrenergic pathways in sneeze-induced urethral continence reflex in rats. Am J Physiol Renal Physiol 2007;292(2):F639-46.
12. Kamo I, Kaiho Y, Canon TW, et al. Functional analysis of active urethral closuremechanisms under sneeze induced stress condition in a rat model of birth trauma. J Urol 2006;176(6 Pt 1):2711-5.
13. Kamo I, Cannon TW, Conway DA, Torimoto K, Chancellor MB, de Groat WC, Yoshimura N. The role of bladder-to-urethral reflexes in urinary continence mechanisms in rats. Am J Physiol Renal Physiol 2004;287(3):F434-41.
14. Strasser H, Ninkovic M, Hess M, Bartsch G, Stenzl A. Anatomic and functional studies of the male and female urethral sphincter. World J Urol 2000;18(5):324-9.
15. Strasser H, Tiefenthaler M, Steinlechner M, Eder I, Bartsch G, Konwalinka G. Age dependent apoptosis and loss of rhabdosphincter cells. J Urol 2000;164(5):1781-5.
16. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284(5411):143-7.
17. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, Mavilio F. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998;279(5356):1528-30.
18. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276(5309):71-4.
19. Dezawa M, Ishikawa H, Itokazu Y, et al. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 2005;309(5732):314-7.
20. Qu Z, Balkir L, van Deutekom JC, Robbins PD, Pruchnic R, Huard J. Development of approaches to improve cell survival in myoblast transfer therapy. J Cell Biol 1998;142(5):1257-67.
21. Oshima H, Payne TR, Urish KL, et al. Differential myocardial infarct repair with muscle stem cells compared to myoblasts. Mol Ther 2005;12(6):1130-41.
22. Lee JY, Paik SY, Yuk SH, Lee JH, Ghil SH, Lee SS. Long term effects of muscle-derived stem cells on leak point pressure and closing pressure in rats with transected pudendal nerves. Mol Cells 2004;18(3):309-13.
23. Kwon D, Kim Y, Pruchnic R, et al. Periurethral cellular injection: comparison of muscle-derived progenitor cells and fibroblasts with regard to efficacy and tissue contractility in an animal model of stress urinary incontinence. Urology 2006;68(2): 449-54.
24. Huard J, Yokoyama T, Pruchnic R, et al. Muscle-derived cell-mediated ex vivo genetherapy for urological dysfunction. Gene Ther 2002;9(23):1617-26.
25. Yokoyama T, Pruchnic R, Lee JY, et al. Autologous primary muscle-derived cells transfer into the lower urinary tract. Tissue Eng 2001;7(4):395-404.
26. Chancellor MB, Yokoyama T, Tirney S, et al. Preliminary results of myoblast injection into the urethra and bladder wall: a possible method for the treatment of stress urinary incontinence and impaired detrusor contractility. Neurourol Urodyn 2000;19(3):279-87.
27. Cannon TW, Lee JY, Somogyi G, Pruchnic R, Smith CP, Huard J, Chancellor MB. Improved sphincter contractility after allogenic muscle-derived progenitor cell injection into the denervated rat urethra. Urology 2003;62(5):958-63.
28. Chermansky CJ, Tarin T, Kwon DD, et al. Intraurethral muscle-derived cell injections increase leak point pressure in a rat model of intrinsic sphincter deficiency. Urology 2004;63(4):780-5.
29. Strasser H, Marksteiner R, Margreiter E, et al. Transurethral ultrasonography-guided injection of adult autologous stem cells versus transurethral endoscopic injection of collagen in treatment of urinary incontinence. World J Urol 2007;25(4):385-92.
30. Carr LK, Steele D, Steele S, et al. 1-year follow-up of autologous muscle-derived stem cell injection pilot study to treat stress urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct 2008;19(6):881-3.
31. Ruberti F, Capsoni S, Comparini A, et al. Phenotypic knockout of nerve growth factor in adult transgenic mice reveals severe deficits in basal forebrain cholinergic neurons, cell death in the spleen, and skeletal muscle dystrophy. J Neurosci 2000; 0(7): 589-601.
32. Rende M, Brizi E, Conner J, et al. Nerve growth factor (NGF) influences differentiation and proliferation of myogenic cells in vitro via TrKA. Int J Dev Neurosci 2000;18(8):869-85.
33. Reddypalli S, Roll K, Lee HK, Lundell M, Barea-Rodriguez E, Wheeler EF. p75NTR-mediated signaling promotes the survival of myoblasts and influences muscle strength. J Cell Physiol 2005;204(3):819-29.
34. Dedkov EI, Kostrominova TY, Borisov AB, Carlson BM. Survival of Schwann cells in chronically denervated skeletal muscles. Acta Neuropathol 2002;103(6):565-74.
35. Neuman M, Zuckerman B, Zilberman M, Farkash A, Beller U. [Tension-free vaginal tape--a new surgical treatment of female urinary stress incontinence]. Harefuah1999;137(5-6):180-2, 264.
36. Nilsson CG, Kuuva N, Falconer C, Rezapour M, Ulmsten U. Long-term results of the tension-free vaginal tape (TVT) procedure for surgical treatment of female stress urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct 2001;12 Suppl 2:S5-8.
37. Jeffry L, Deval B, Birsan A, Soriano D, Darai E. Objective and subjective cure rates after tension-free vaginal tape for treatment of urinary incontinence. Urology 2001;58(5):702-6.
38. Ward KL, Hilton P. A prospective multicenter randomized trial of tension-free vaginal tape and colposuspension for primary urodynamic stress incontinence: two-year follow-up. Am J Obstet Gynecol 2004;190(2):324-31.
1. Kwon D, Kim Y, Pruchnic R, et al. Periurethral cellular injection: comparison of muscle-derived progenitor cells and fibroblasts with regard to efficacy and tissue contractility in an animal model of stress urinary incontinence. Urology 2006;68(2):449-54.
2. Sato K, Li Y, Foster W, et al. Improvement of muscle healing through enhancement of muscle regeneration and prevention of fibrosis. Muscle Nerve 2003;28(3):365-72.
3. Menetrey J, Kasemkijwattana C, Fu FH, Moreland MS, Huard J. Suturing versus immobilization of a muscle laceration. A morphological and functional study in a mouse model. Am J Sports Med 1999;27(2):222-9.
4. Li Y, Li J, Zhu J, et al. Decorin gene transfer promotes muscle cell differentiation and muscle regeneration. Mol Ther 2007;15(9):1616-22.
5. Ono A, Utsugi M, Masubuchi K, et al. Glutathione redox regulates TGF-beta-induced fibrogenic effects through Smad3 activation. FEBS Lett 2009;583(2):357-62.
6. Zhao J, Shi W, Wang YL, et al. Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol 2002;282(3):L585-93.
7. Liu D, Black BL, Derynck R. TGF-βinhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3. Genes Dev 2001;15(22): 2950-66.
8. Liu D, Kang JS, Derynck R. TGF-beta-activated Smad3 represses MEF2-dependent transcription in myogenic differentiation. EMBO J 2004;23(7):1557-66.
9. Qu-Petersen Z, Deasy B, Jankowski R, et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol 2002;157(5):851-64.
10. Zhang H, Feng W, Liao W, Ma X, Han Q, Zhang Y. The gp130/STAT3 signaling pathway mediates beta-adrenergic receptor-induced atrial natriuretic factor expression in cardiomyocytes. FEBS J 2008;275(14):3590-7.
11. Lee JY, Paik SY, Yuk SH, Lee JH, Ghil SH, Lee SS. Long term effects of muscle-derived stem cells on leak point pressure and closing pressure in rats withtransected pudendal nerves. Mol Cells 2004;18(3):309-13.
12. Ward KL, Hilton P. A prospective multicenter randomized trial of tension-free vaginal tape and colposuspension for primary urodynamic stress incontinence: two-year follow-up. Am J Obstet Gynecol 2004;190(2):324-31.
13. Gharaibeh B, Lu A, Tebbets J, et al. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc 2008;3(9):1501-9.
14. Reiser J, Zhang XY, Hemenway CS, Mondal D, Pradhan L, La Russa VF. Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases. Expert Opin Biol Ther 2005;5(12):1571-84.
15. Van Damme A, Thorrez L, Ma L, et al. Efficient lentiviral transduction and improved engraftment of human bone marrow mesenchymal cells. Stem Cells 2006;24(4): 896-907.
16. Choi WY, Giraldez AJ, Schier AF. Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430. Science 2007;318(5848):271-4.
17. Kato M, Zhang J, Wang M, Lanting L, Yuan H, Rossi JJ, Natarajan R. MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-beta-induced collagen expression via inhibition of E-box repressors. Proc Natl Acad Sci U S A 2007;104(9):3432-7.
18. Dooley S, Delvoux B, Lahme B, Mangasser-Stephan K, Gressner AM. Modulation of transforming growth factor beta response and signaling during transdifferentiation of rat hepatic stellate cells to myofibroblasts. Hepatology 2000;31(5):1094-106.
19. Li Y, Huard J. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. Am J Pathol 2002;161(3):895-907.
2. Hunskaar S, Lose G, Sykes D, Voss S. The prevalence of urinary incontinence in women in four European countries. BJU Int 2004;93(3):324-30.
3. Hunskaar S, Burgio K, Diokno A, Herzog AR, Hjalmas K, Lapitan MC. Epidemiology and natural history of urinary incontinence in women. Urology 2003;62(4 Suppl 1):16-23.
4. Miller DC, Saigal CS, Litwin MS. The demographic burden of urologic diseases in America. Urol Clin North Am 2009;36(1):11-27, v.
5. NIH state-of-the-science conference statement on prevention of fecal and urinary incontinence in adults. NIH Consens State Sci Statements 2007;24(1):1-37.
6. Farage MA, Miller KW, Berardesca E, Maibach HI. Psychosocial and societal burden of incontinence in the aged population: a review. Arch Gynecol Obstet 2008;277(4):285-90.
7. Kevorkian R. Physiology of incontinence. Clin Geriatr Med 2004;20(3):409-25, v.
8. Takahashi S, Homma Y, Fujishiro T, Hosaka Y, Kitamura T, Kawabe K. Electromyographic study of the striated urethral sphincter in type 3 stress incontinence: evidence of myogenic-dominant damages. Urology 2000;56(6):946-50.
9. Hasiam J. Current treatments for patients with stress urinary incontinence. Nurs Times 2004;100(2):50-1.
10. Strasser H, Marksteiner R, Margreiter E, et al. [Stem cell therapy for urinary incontinence]. Urologe A 2004;43(10):1237-41.
11. Mitterberger M, Pinggera GM, Marksteiner R, et al. Adult stem cell therapy of female stress urinary incontinence. Eur Urol 2008;53(1):169-75.
12. Cao B, Huard J. Muscle-derived stem cells. Cell Cycle 2004;3(2):104-7.
13. Charge SB, Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev 2004;84(1):209-38.
14. Pagani FD, DerSimonian H, Zawadzka A, et al. Autologous skeletal myoblaststransplanted to ischemia-damaged myocardium in humans. Histological analysis of cell survival and differentiation. J Am Coll Cardiol 2003;41(5):879-88.
15. Herreros J, Prosper F, Perez A, et al. Autologous intramyocardial injection of cultured skeletal muscle-derived stem cells in patients with non-acute myocardial infarction. Eur Heart J 2003;24(22):2012-20.
16. Howell JC, Lee WH, Morrison P, Zhong J, Yoder MC, Srour EF. Pluripotent stem cells identified in multiple murine tissues. Ann N Y Acad Sci 2003;996:158-73.
17. Cannon TW, Lee JY, Somogyi G, Pruchnic R, Smith CP, Huard J, Chancellor MB. Improved sphincter contractility after allogenic muscle-derived progenitor cell injection into the denervated rat urethra. Urology 2003;62(5):958-63.
18. Lee JY, Paik SY, Yuk SH, Lee JH, Ghil SH, Lee SS. Long term effects of muscle-derived stem cells on leak point pressure and closing pressure in rats with transected pudendal nerves. Mol Cells 2004;18(3):309-13.
19. Chermansky CJ, Tarin T, Kwon DD, et al. Intraurethral muscle-derived cell injections increase leak point pressure in a rat model of intrinsic sphincter deficiency. Urology 2004;63(4):780-5.
20. Lee JY, Cannon TW, Pruchnic R, Fraser MO, Huard J, Chancellor MB. The effects of periurethral muscle-derived stem cell injection on leak point pressure in a rat model of stress urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct 2003;14(1):31-7; discussion 37.
21. Liu D, Black BL, Derynck R. TGF-βinhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3. Genes Dev 2001;15(22):2950-66.
22. Liu D, Kang JS, Derynck R. TGF-beta-activated Smad3 represses MEF2-dependent transcription in myogenic differentiation. EMBO J 2004;23(7):1557-66.
23. Li Y, Foster W, Deasy BM, et al. Transforming growth factor-beta1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis. Am J Pathol 2004;164(3):1007-19.
24. Kwon D, Kim Y, Pruchnic R, et al. Periurethral cellular injection: comparison of muscle-derived progenitor cells and fibroblasts with regard to efficacy and tissue contractility in an animal model of stress urinary incontinence. Urology 2006;68(2):449-54.
25. Chamberlain JR, Schwarze U, Wang PR, et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 2004;303(5661):1198-201.
26. Qu-Petersen Z, Deasy B, Jankowski R, et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol 2002;157(5):851-64.
27. Lee JY, Qu-Petersen Z, Cao B, et al. Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing. J Cell Biol 2000;150(5):1085-100.
28. Gharaibeh B, Lu A, Tebbets J, et al. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc 2008;3(9):1501-9.
29.姚启盛,叶章群,陈丛波,王小康,王卫民,陈立新. Dispase在成年鼠骨骼肌干细胞纯化中的价值.郧阳医学院学报; 2004:336-337.
30. Huard J, Cao B, Qu-Petersen Z. Muscle-derived stem cells: potential for muscle regeneration. Birth Defects Res C Embryo Today 2003;69(3):230-7.
31. Li Y, Huard J. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. Am J Pathol 2002;161(3):895-907.
32. Zheng B, Cao B, Crisan M, et al. Prospective identification of myogenic endothelial cells in human skeletal muscle. Nat Biotechnol 2007;25(9):1025-34.
33. Lu A, Cummins JH, Pollett JB, Cao B, Sun B, Rudnicki MA, Huard J. Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics. Gene Ther 2008;15(15):1116-25.
34. Jankowski RJ, Haluszczak C, Trucco M, Huard J. Flow cytometric characterization of myogenic cell populations obtained via the preplate technique: potential for rapid isolation of muscle-derived stem cells. Hum Gene Ther 2001;12(6):619-28.
35. Deasy BM, Lu A, Tebbets JC, et al. A role for cell sex in stem cell-mediated skeletal muscle regeneration: female cells have higher muscle regeneration efficiency. J Cell Biol 2007;177(1):73-86.
36. Roberts AB, Russo A, Felici A, Flanders KC. Smad3: a key player in pathogenetic mechanisms dependent on TGF-beta. Ann N Y Acad Sci 2003;995:1-10.
37. Flanders KC, Sullivan CD, Fujii M, et al. Mice lacking Smad3 are protected againstcutaneous injury induced by ionizing radiation. Am J Pathol 2002;160(3):1057-68.
38. Danisovic L, Varga I, Polak S, Ulicna M, Bohmer D, Vojtassak J. Morphology of in vitro expanded human muscle-derived stem cells. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2008;152(2):235-8.
39. Schnaper HW, Hayashida T, Hubchak SC, Poncelet AC. TGF-βsignal transduction and mesangial cell fibrogenesis. Am J Physiol Renal Physiol 2003;284(2):F243-52.
40. Verrecchia F, Mauviel A. Transforming growth factor-beta and fibrosis. World J Gastroenterol 2007;13(22):3056-62.
41. Verrecchia F, Laboureau J, Verola O, et al. Skin involvement in scleroderma--where histological and clinical scores meet. Rheumatology (Oxford) 2007;46(5):833-41.
42. Shi Y, Massague J. Mechanisms of TGF-βsignaling from cell membrane to the nucleus. Cell 2003;113(6):685-700.
43. Attisano L, Wrana JL. Smads as transcriptional co-modulators. Curr Opin Cell Biol 2000;12(2):235-43.
44. Chen W, Fu X, Sheng Z. Review of current progress in the structure and function of Smad proteins. Chin Med J (Engl) 2002;115(3):446-50.
45. Scherr M, Eder M. Gene transfer into hematopoietic stem cells using lentiviral vectors. Curr Gene Ther 2002;2(1):45-55.
46. Sato M, Markiewicz M, Yamanaka M, et al. Modulation of transforming growth factor-beta (TGF-beta) signaling by endogenous sphingolipid mediators. J Biol Chem 2003;278(11):9276-82.
47. Verrecchia F, Chu ML, Mauviel A. Identification of novel TGF-β/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach. J Biol Chem 2001;276(20):17058-62.
48. Zhao J, Shi W, Wang YL, et al. Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol 2002;282(3): L585-93.
49. Schnabl B, Kweon YO, Frederick JP, Wang XF, Rippe RA, Brenner DA. The role of Smad3 in mediating mouse hepatic stellate cell activation. Hepatology 2001;34(1):89-100.
50. Fujimoto M, Maezawa Y, Yokote K, et al. Mice lacking Smad3 are protected againststreptozotocin-induced diabetic glomerulopathy. Biochem Biophys Res Commun 2003;305(4):1002-7.
51. Scherr M, Battmer K, Blomer U, Schiedlmeier B, Ganser A, Grez M, Eder M. Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells. Blood 2002;99(2):709-12.
52. Lan HY, Mu W, Tomita N, et al. Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model. J Am Soc Nephrol 2003;14(6):1535-48.
53. Terada Y, Hanada S, Nakao A, Kuwahara M, Sasaki S, Marumo F. Gene transfer of Smad7 using electroporation of adenovirus prevents renal fibrosis in post-obstructed kidney. Kidney Int 2002;61(1 Suppl):S94-8.
54. Nakao A, Fujii M, Matsumura R, Kumano K, Saito Y, Miyazono K, Iwamoto I. Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice. J Clin Invest 1999;104(1):5-11.
55. Pines M, Snyder D, Yarkoni S, Nagler A. Halofuginone to treat fibrosis in chronic graft-versus-host disease and scleroderma. Biol Blood Marrow Transplant 2003;9(7):417-25.
56. Bruck R, Genina O, Aeed H, Alexiev R, Nagler A, Avni Y, Pines M. Halofuginone to prevent and treat thioacetamide-induced liver fibrosis in rats. Hepatology 2001;33(2):379-86.
57. McGaha TL, Phelps RG, Spiera H, Bona C. Halofuginone, an inhibitor of type-I collagen synthesis and skin sclerosis, blocks transforming-growth-factor-beta-mediated Smad3 activation in fibroblasts. J Invest Dermatol 2002;118(3):461-70.
58. Nishitsuji H, Ikeda T, Miyoshi H, Ohashi T, Kannagi M, Masuda T. Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary non-dividing cells. Microbes Infect 2004;6(1):76-85.
59. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296(5567):550-3.
60. Ono A, Utsugi M, Masubuchi K, et al. Glutathione redox regulates TGF-beta-induced fibrogenic effects through Smad3 activation. FEBS Lett 2009;583(2):357-62.
61. Holterman CE, Rudnicki MA. Molecular regulation of satellite cell function. Semin Cell Dev Biol 2005;16(4-5):575-84.
62. Cornelison DD, Filla MS, Stanley HM, Rapraeger AC, Olwin BB. Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration. Dev Biol 2001;239(1):79-94.
63.徐惠成,王延洲,等.肌源性干细胞治疗压力性尿失禁的实验研究.第三军医大学学报2007年02期; 2007:113-5.
64. Huard J, Yokoyama T, Pruchnic R, et al. Muscle-derived cell-mediated ex vivo gene therapy for urological dysfunction. Gene Ther 2002;9(23):1617-26.
65. Yokoyama T, Pruchnic R, Lee JY, et al. Autologous primary muscle-derived cells transfer into the lower urinary tract. Tissue Eng 2001;7(4):395-404.
66. Chancellor MB, Yokoyama T, Tirney S, et al. Preliminary results of myoblast injection into the urethra and bladder wall: a possible method for the treatment of stress urinary incontinence and impaired detrusor contractility. Neurourol Urodyn 2000;19(3): 279-87.
1. Hannestad YS, Rortveit G, Sandvik H, Hunskaar S. A community-based epidemiological survey of female urinary incontinence: the Norwegian EPINCONT study. Epidemiology of Incontinence in the County of Nord-Trondelag. J Clin Epidemiol 2000;53(11):1150-7.
2. Subak LL, Whitcomb E, Shen H, Saxton J, Vittinghoff E, Brown JS. Weight loss: a novel and effective treatment for urinary incontinence. J Urol 2005;174(1):190-5.
3. Swithinbank L, Hashim H, Abrams P. The effect of fluid intake on urinary symptoms in women. J Urol 2005;174(1):187-9.
4. Hannestad YS, Rortveit G, Daltveit AK, Hunskaar S. Are smoking and other lifestyle factors associated with female urinary incontinence? The Norwegian EPINCONT Study. BJOG 2003;110(3):247-54.
5. Bo K, Borgen JS. Prevalence of stress and urge urinary incontinence in elite athletes and controls. Med Sci Sports Exerc 2001;33(11):1797-802.
6. Richter HE, Burgio KL, Goode PS, et al. Non-surgical management of stress urinary incontinence: ambulatory treatments for leakage associated with stress (ATLAS) trial. Clin Trials 2007;4(1):92-101.
7. Hay-Smith EJ, Bo Berghmans LC, Hendriks HJ, de Bie RA, van Waalwijk van Doorn ES. Pelvic floor muscle training for urinary incontinence in women. Cochrane Database Syst Rev 2001(1):CD001407.
8. Dumoulin C, Hay-Smith J. Pelvic floor muscle training versus no treatment for urinary incontinence in women. A Cochrane systematic review. Eur J Phys Rehabil Med 2008;44(1):47-63.
9. Aksac B, Aki S, Karan A, Yalcin O, Isikoglu M, Eskiyurt N. Biofeedback and pelvic floor exercises for the rehabilitation of urinary stress incontinence. Gynecol ObstetInvest 2003;56(1):23-7.
10. Spruijt J, Vierhout M, Verstraeten R, Janssens J, Burger C. Vaginal electrical stimulation of the pelvic floor: a randomized feasibility study in urinary incontinent elderly women. Acta Obstet Gynecol Scand 2003;82(11):1043-8.
11. Alhasso A, Glazener CM, Pickard R, N'dow J. Adrenergic drugs for urinary incontinence in adults. Cochrane Database Syst Rev 2005(3):CD001842.
12. Boy S, Reitz A, Wirth B, Knapp PA, Braun PM, Haferkamp A, Schurch B. Facilitatory neuromodulative effect of duloxetine on pudendal motor neurons controlling the urethral pressure: a functional urodynamic study in healthy women. Eur Urol 2006;50(1):119-25.
13. Athanasiou S, Chaliha C, Digesu GA, Sotiropoulou M, Georgoulias N, Khullar V, Antsaklis A. The effects of duloxetine on urethral function and sphincter morphology. Int Urogynecol J Pelvic Floor Dysfunct 2007;18(7):763-7.
14. Lin AT, Sun MJ, Tai HL, et al. Duloxetine versus placebo for the treatment of women with stress predominant urinary incontinence in Taiwan: a double-blind, randomized, placebo-controlled trial. BMC Urol 2008;8:2.
15. Cardozo L, Drutz HP, Baygani SK, Bump RC. Pharmacological treatment of women awaiting surgery for stress urinary incontinence. Obstet Gynecol 2004;104(3):511-9.
16. Duckett J, Patil A, Aggarwal I. The effect of duloxetine on urethral sphincter morphology. Ultrasound Obstet Gynecol 2008;31(2):206-9.
17. Ghoniem GM, Van Leeuwen JS, Elser DM, Freeman RM, Zhao YD, Yalcin I, Bump RC. A randomized controlled trial of duloxetine alone, pelvic floor muscle training alone, combined treatment and no active treatment in women with stress urinary incontinence. J Urol 2005;173(5):1647-53.
18. Hurley DJ, Turner CL, Yalcin I, Viktrup L, Baygani SK. Duloxetine for the treatment of stress urinary incontinence in women: an integrated analysis of safety. Eur J Obstet Gynecol Reprod Biol 2006;125(1):120-8.
19. Mitterberger M, Pinggera GM, Marksteiner R, et al. Adult stem cell therapy of female stress urinary incontinence. Eur Urol 2008;53(1):169-75.
20. Duckett JR, Vella M, Kavalakuntla G, Basu M. Tolerability and efficacy of duloxetine in a nontrial situation. BJOG 2007;114(5):543-7.
21. Vella M, Duckett J, Basu M. Duloxetine 1 year on: the long-term outcome of a cohort of women prescribed duloxetine. Int Urogynecol J Pelvic Floor Dysfunct 2008;19(7):961-4.
22. Basu M, Duckett J. The treatment of urinary incontinence with Duloxetine. J Obstet Gynaecol 2008;28(2):166-9.
23. Grady D, Brown JS, Vittinghoff E, Applegate W, Varner E, Snyder T. Postmenopausal hormones and incontinence: the Heart and Estrogen/Progestin Replacement Study. Obstet Gynecol 2001;97(1):116-20.
24. Latthe PM, Foon R, Khan K. Nonsurgical treatment of stress urinary incontinence (SUI): grading of evidence in systematic reviews. BJOG 2008;115(4):435-44.
25. Castro-Diaz D, Amoros MA. Pharmacotherapy for stress urinary incontinence. Curr Opin Urol 2005;15(4):227-30.
26. Schagen van Leeuwen JH, Lange RR, Jonasson AF, Chen WJ, Viktrup L. Efficacy and safety of duloxetine in elderly women with stress urinary incontinence or stress-predominant mixed urinary incontinence. Maturitas 2008;60(2):138-47.
1. Corcos J, Beaulieu S, Donovan J, Naughton M, Gotoh M. Quality of life assessment in men and women with urinary incontinence. J Urol 2002;168(3):896-905.
2. Norton P, Brubaker L. Urinary incontinence in women. Lancet 2006;367(9504):57-67.
3. Meyer S, Schreyer A, De Grandi P, Hohlfeld P. The effects of birth on urinary continence mechanisms and other pelvic-floor characteristics. Obstet Gynecol 1998;92(4 Pt 1):613-8.
4. Radley SC, Chapple CR, Bryan NP, Clarke DE, Craig DA. Effect of methoxamine on maximum urethral pressure in women with genuine stress incontinence: a placebo- controlled, double-blind crossover study. Neurourol Urodyn 2001;20(1):43-52.
5. Viktrup L, Perahia DG, Tylee A. Duloxetine Treatment of Stress Urinary Incontinence in Women Does Not Induce Mania or Hypomania. Prim Care Companion J Clin Psychiatry 2004;6(6):239-243.
6. Dewan PA, Byard RW. Re: Transurethral Polytef injection for post-prostatectomy urinary incontinence and Long-term follow-up of women treated with periurethral Teflon injections for stress incontinence. Br J Urol 1993;71(1):112.
7. Bent AE, Tutrone RT, McLennan MT, Lloyd LK, Kennelly MJ, Badlani G. Treatment of intrinsic sphincter deficiency using autologous ear chondrocytes as a bulking agent. Neurourol Urodyn 2001;20(2):157-65.
8. Lee JY, Cannon TW, Pruchnic R, Fraser MO, Huard J, Chancellor MB. The effects of periurethral muscle-derived stem cell injection on leak point pressure in a rat model of stress urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct 2003;14(1):31-7; discussion 37.
9. DeLancey JO. Structural support of the urethra as it relates to stress urinary incontinence: the hammock hypothesis. Am J Obstet Gynecol 1994;170(6):1713-20; discussion 1720-3.
10. de Groat WC. Integrative control of the lower urinary tract: preclinical perspective. Br J Pharmacol 2006;147 Suppl 2:S25-40.
11. Kaiho Y, Kamo I, Chancellor MB, Arai Y, de Groat WC, Yoshimura N. Role of noradrenergic pathways in sneeze-induced urethral continence reflex in rats. Am J Physiol Renal Physiol 2007;292(2):F639-46.
12. Kamo I, Kaiho Y, Canon TW, et al. Functional analysis of active urethral closuremechanisms under sneeze induced stress condition in a rat model of birth trauma. J Urol 2006;176(6 Pt 1):2711-5.
13. Kamo I, Cannon TW, Conway DA, Torimoto K, Chancellor MB, de Groat WC, Yoshimura N. The role of bladder-to-urethral reflexes in urinary continence mechanisms in rats. Am J Physiol Renal Physiol 2004;287(3):F434-41.
14. Strasser H, Ninkovic M, Hess M, Bartsch G, Stenzl A. Anatomic and functional studies of the male and female urethral sphincter. World J Urol 2000;18(5):324-9.
15. Strasser H, Tiefenthaler M, Steinlechner M, Eder I, Bartsch G, Konwalinka G. Age dependent apoptosis and loss of rhabdosphincter cells. J Urol 2000;164(5):1781-5.
16. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284(5411):143-7.
17. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, Mavilio F. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998;279(5356):1528-30.
18. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276(5309):71-4.
19. Dezawa M, Ishikawa H, Itokazu Y, et al. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 2005;309(5732):314-7.
20. Qu Z, Balkir L, van Deutekom JC, Robbins PD, Pruchnic R, Huard J. Development of approaches to improve cell survival in myoblast transfer therapy. J Cell Biol 1998;142(5):1257-67.
21. Oshima H, Payne TR, Urish KL, et al. Differential myocardial infarct repair with muscle stem cells compared to myoblasts. Mol Ther 2005;12(6):1130-41.
22. Lee JY, Paik SY, Yuk SH, Lee JH, Ghil SH, Lee SS. Long term effects of muscle-derived stem cells on leak point pressure and closing pressure in rats with transected pudendal nerves. Mol Cells 2004;18(3):309-13.
23. Kwon D, Kim Y, Pruchnic R, et al. Periurethral cellular injection: comparison of muscle-derived progenitor cells and fibroblasts with regard to efficacy and tissue contractility in an animal model of stress urinary incontinence. Urology 2006;68(2): 449-54.
24. Huard J, Yokoyama T, Pruchnic R, et al. Muscle-derived cell-mediated ex vivo genetherapy for urological dysfunction. Gene Ther 2002;9(23):1617-26.
25. Yokoyama T, Pruchnic R, Lee JY, et al. Autologous primary muscle-derived cells transfer into the lower urinary tract. Tissue Eng 2001;7(4):395-404.
26. Chancellor MB, Yokoyama T, Tirney S, et al. Preliminary results of myoblast injection into the urethra and bladder wall: a possible method for the treatment of stress urinary incontinence and impaired detrusor contractility. Neurourol Urodyn 2000;19(3):279-87.
27. Cannon TW, Lee JY, Somogyi G, Pruchnic R, Smith CP, Huard J, Chancellor MB. Improved sphincter contractility after allogenic muscle-derived progenitor cell injection into the denervated rat urethra. Urology 2003;62(5):958-63.
28. Chermansky CJ, Tarin T, Kwon DD, et al. Intraurethral muscle-derived cell injections increase leak point pressure in a rat model of intrinsic sphincter deficiency. Urology 2004;63(4):780-5.
29. Strasser H, Marksteiner R, Margreiter E, et al. Transurethral ultrasonography-guided injection of adult autologous stem cells versus transurethral endoscopic injection of collagen in treatment of urinary incontinence. World J Urol 2007;25(4):385-92.
30. Carr LK, Steele D, Steele S, et al. 1-year follow-up of autologous muscle-derived stem cell injection pilot study to treat stress urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct 2008;19(6):881-3.
31. Ruberti F, Capsoni S, Comparini A, et al. Phenotypic knockout of nerve growth factor in adult transgenic mice reveals severe deficits in basal forebrain cholinergic neurons, cell death in the spleen, and skeletal muscle dystrophy. J Neurosci 2000; 0(7): 589-601.
32. Rende M, Brizi E, Conner J, et al. Nerve growth factor (NGF) influences differentiation and proliferation of myogenic cells in vitro via TrKA. Int J Dev Neurosci 2000;18(8):869-85.
33. Reddypalli S, Roll K, Lee HK, Lundell M, Barea-Rodriguez E, Wheeler EF. p75NTR-mediated signaling promotes the survival of myoblasts and influences muscle strength. J Cell Physiol 2005;204(3):819-29.
34. Dedkov EI, Kostrominova TY, Borisov AB, Carlson BM. Survival of Schwann cells in chronically denervated skeletal muscles. Acta Neuropathol 2002;103(6):565-74.
35. Neuman M, Zuckerman B, Zilberman M, Farkash A, Beller U. [Tension-free vaginal tape--a new surgical treatment of female urinary stress incontinence]. Harefuah1999;137(5-6):180-2, 264.
36. Nilsson CG, Kuuva N, Falconer C, Rezapour M, Ulmsten U. Long-term results of the tension-free vaginal tape (TVT) procedure for surgical treatment of female stress urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct 2001;12 Suppl 2:S5-8.
37. Jeffry L, Deval B, Birsan A, Soriano D, Darai E. Objective and subjective cure rates after tension-free vaginal tape for treatment of urinary incontinence. Urology 2001;58(5):702-6.
38. Ward KL, Hilton P. A prospective multicenter randomized trial of tension-free vaginal tape and colposuspension for primary urodynamic stress incontinence: two-year follow-up. Am J Obstet Gynecol 2004;190(2):324-31.
1. Kwon D, Kim Y, Pruchnic R, et al. Periurethral cellular injection: comparison of muscle-derived progenitor cells and fibroblasts with regard to efficacy and tissue contractility in an animal model of stress urinary incontinence. Urology 2006;68(2):449-54.
2. Sato K, Li Y, Foster W, et al. Improvement of muscle healing through enhancement of muscle regeneration and prevention of fibrosis. Muscle Nerve 2003;28(3):365-72.
3. Menetrey J, Kasemkijwattana C, Fu FH, Moreland MS, Huard J. Suturing versus immobilization of a muscle laceration. A morphological and functional study in a mouse model. Am J Sports Med 1999;27(2):222-9.
4. Li Y, Li J, Zhu J, et al. Decorin gene transfer promotes muscle cell differentiation and muscle regeneration. Mol Ther 2007;15(9):1616-22.
5. Ono A, Utsugi M, Masubuchi K, et al. Glutathione redox regulates TGF-beta-induced fibrogenic effects through Smad3 activation. FEBS Lett 2009;583(2):357-62.
6. Zhao J, Shi W, Wang YL, et al. Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol 2002;282(3):L585-93.
7. Liu D, Black BL, Derynck R. TGF-βinhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3. Genes Dev 2001;15(22): 2950-66.
8. Liu D, Kang JS, Derynck R. TGF-beta-activated Smad3 represses MEF2-dependent transcription in myogenic differentiation. EMBO J 2004;23(7):1557-66.
9. Qu-Petersen Z, Deasy B, Jankowski R, et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol 2002;157(5):851-64.
10. Zhang H, Feng W, Liao W, Ma X, Han Q, Zhang Y. The gp130/STAT3 signaling pathway mediates beta-adrenergic receptor-induced atrial natriuretic factor expression in cardiomyocytes. FEBS J 2008;275(14):3590-7.
11. Lee JY, Paik SY, Yuk SH, Lee JH, Ghil SH, Lee SS. Long term effects of muscle-derived stem cells on leak point pressure and closing pressure in rats withtransected pudendal nerves. Mol Cells 2004;18(3):309-13.
12. Ward KL, Hilton P. A prospective multicenter randomized trial of tension-free vaginal tape and colposuspension for primary urodynamic stress incontinence: two-year follow-up. Am J Obstet Gynecol 2004;190(2):324-31.
13. Gharaibeh B, Lu A, Tebbets J, et al. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc 2008;3(9):1501-9.
14. Reiser J, Zhang XY, Hemenway CS, Mondal D, Pradhan L, La Russa VF. Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases. Expert Opin Biol Ther 2005;5(12):1571-84.
15. Van Damme A, Thorrez L, Ma L, et al. Efficient lentiviral transduction and improved engraftment of human bone marrow mesenchymal cells. Stem Cells 2006;24(4): 896-907.
16. Choi WY, Giraldez AJ, Schier AF. Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430. Science 2007;318(5848):271-4.
17. Kato M, Zhang J, Wang M, Lanting L, Yuan H, Rossi JJ, Natarajan R. MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-beta-induced collagen expression via inhibition of E-box repressors. Proc Natl Acad Sci U S A 2007;104(9):3432-7.
18. Dooley S, Delvoux B, Lahme B, Mangasser-Stephan K, Gressner AM. Modulation of transforming growth factor beta response and signaling during transdifferentiation of rat hepatic stellate cells to myofibroblasts. Hepatology 2000;31(5):1094-106.
19. Li Y, Huard J. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. Am J Pathol 2002;161(3):895-907.